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Age-dependent enzyme changes in Drosophila melanogaster
Exp. Geront. VoL 4. pp. 207-222, Pergamon Press 1969. Printed in Great Britain
A G E - D E P E N D E N T ENZYME CHANGES IN DROSOPHILA MELANOGASTER* J. C. HALL Department of Genetics, University of Washington, Seattle, Washington 98105
(Received 29 May 1969)
INTRODUCTION THE PURPOSEof this investigation is to seek ways of examining the involvement of the genome of Drosophila melanogaster in the process of senescence. Genetic causes of aging may be of two kinds: (1) built-in errors or contradictions of design (faults of the genotype as established at the time of fertilization); and (2) somatic mutations (microaccidents which disrupt the structure of genes or chromosomes). Evidence bearing on somatic mutation theories of aging (such as that of Szilard, 1959) is in some cases consistent, but in others so contradictory that one can say, at least, that it is improbable that aging in general is a consequence of the accumulation with time of somatic gene mutations or chromosome aberrations. To examine the first possibility--a genetic determination of senescence--it would be desirable to search for, and characterize, mutations that affect aging. Gonzalez (1923) demonstrated, in D. melanogaster, that abnormalities in structure caused by the presence of homozygous mutant genes (such as purple eye color) cause a decrease in longevity. But a study of such mutants would say little about senescence, for the only connection they have with aging is that they lead to early death. The kind of inherited faults which could be termed true aging mutations would be genetic lesions whose phenotype is a mimicry of aging based on a definite set of criteria of the phenomenon in addition to decreased lifespan. Casarett (1964) has put forth criteria which can be used to detect a mutation whose phenotype is premature aging. A population of individuals carrying this putative aging mutation would show (1) an earlier increase in mortality without alteration of the shape of the mortality curve; (2) a proportional advancement in time of all diseases or causes of death; (3) a proportional advancement in time of all morphological and physiological manifestations of the aging process. There are a few mutations known which seem to result in a form of premature senescence. Werner's syndrome in man is inherited as a single autosomal recessive mutation (Epstein, Martin, Schultz and Motulsky, 1966). Many (though not all) of the symptoms of this condition, which first appear at about age twenty in individuals homozygous for the lesion, are quite similar to features manifested in normal human senescence. But Epstein et al. (1966) are very cautious as to whether this disease is truly precocious or accelerated senescence, or whether it may better be considered a "caricature" of aging. * Research sponsored jointly by Grant 5 T01 GM 00182 and Grant RG-9965, both from the U.S. Public Health Service. 207
208
j.C. H&LL
In any event, man is in many ways a refractory organism with which to study the the nature of a genetic alteration. Drosophila melanogaster, on the other hand, has a powerful genetic system; the advantage that many individuals can be screened for a putative aging mutation; and the property that survival curves for flies carrying the putative mutation can easily be determined with enough organisms to make the shapes of the curves meaningful. However, very little is known of the diseases or causes of death in normal Drosophila, or of the morphological and physiological features of aging. The purpose of this study, then, is to determine some of the symptoms of senescence in fruit flies; that is, to describe changes which can be consistently found to occur over the life of the organism. Against such a background, a search for inherited changes which alter the aging process could be carried out. Rockstein (1966) has found that there are changes in the activities of certain enzymes which can be demonstrated in aging male house flies (Musca domestica L). The plan of the present investigation is to ask whether enzyme changes---of a qualitative and quantitative nature---occur in aging Drosophila melanogaster. It has been found that of four enzymes examined (two by electrophoresis, two by a direct measure of activity), all show changes as a function of the age of the flies from which they were extracted. These enzymes, moreover, were not chosen because of any a priori suspicion that they were especially related to senescence. METHODS AND PROCEDURE
Survival curves The Drosophila melanogaster stock used in these experiments was a Canton-S wild type. Virgin females and males whose age varied from 0 to 12 hours were collected from bottle cultures. Four separate experiments were begun at the time of collection: (A) ten females per vial (240 vials), (B) ten males per vial (240 vials), (C) five females and five males per vial (240 vials), and (D) one female and one male per vial (120 vials). Also at this time a large number of excess females and males were quick-frozen in an ethanol plus dry ice bath and stored at - 60°C. At 72-hr intervals, the flies in the four experiments were transferred to fresh vials, with the number of survivors being noted at each transfer. In addition, the old cultures in experiments (C) and (D) were saved so that they could be scored later for the presence or absence of progeny. On the 25th, the 49th, the 58th, and the 67th day from the beginning of the experiments, 45-55 flies from experiments (A) and (B) were etherized, quick-frozen, and stored as before, to be used for the enzyme assays.
Assays of hexose-P-isomerase and esterase These two enzymes were assayed by starch gel electrophoresis to see if an altered banding pattern would appear as a function of the age oftheflies. Frozen flies were singly ground with a glass rod in 10 × 75 mm glass test tubes containing 0.1 ml 0.05 M PO~ buffer, pH 6.5. Starch gel electrophoresis was done according to the method of Smithies (1955). Each gel was made with 44.0 g starch (Electrostarch Company, Madison, Wisconsin) in 400 ml 0"005 M PO4, pH 7"0. The buffer in the gel boxes was 0.1 M PO~, pH 7-0. Paper inserts (a double thickness of Whatman No. 3 filter paper) soaked in the crude fly extracts were placed in the gel, and a potential difference of 10.2 V/cm was applied across the length of the gel for 4 hr. To normalize the distances traveled by the
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A M E L A N O G A S T E R
209
enzymes from gel to gel, an insert soaked in "reference extract" (15 0.5 day-old males or females ground in 1.5 ml buffer) was placed in each gel alongside the experimental inserts. When assaying extracts of the old flies, two of the twenty inserts in each gel were soaked in extracts from the 0.5 day-old flies; a direct comparison of migration and staining intensity of the enzymes from old flies vs. those from young flies was thus possible. At the conclusion of a run, the gel was sliced in half and the top half was stained by the method of Chapman, Hennessey, Waltersdorph, Huennekens, and Garbio (1962) for hexose-P-isomerase activity. The reaction mixture was modified to be as follows: 5-0 ml 0.3 M Tris buffer, pH 8.0 + 0.5 ml 0.1 M Mg 9"+ + 0.2 ml 10 mg/ml D-fructose-6phosphate disodium salt (Sigma Chemical Company, St. Louis, Missouri) + 0.2 ml 6 mg/ml NADP + (Sigma) + 0.3 ml 1 U/ml glucose-6-phosphate dehydrogenase (Calbiochem, Los Angeles, California) + 1 mg phenazine methosulfate (Sigma) + 1 mg M T T tetrazolium (Sigma) + 10 per cent agar (dissolved in hot distilled water then cooled to less than 45°C). The bottom half of the gel was stained after the method of Harris, Hopkinson, and Robson (1962) for esterase activity. The reaction mixture consisted of (0.02 g oc naphthylacetate (Sigma) in 1 ml H20 + 1 ml acetone) + 100 ml 0-2 M PO~, pH 6.8 + 40 mg fast red TR salt (Sigma). As a preliminary experiment, a control gel was run in which were inserted extracts from frozen 0.5 day-old males and females, and extracts from fresh (just-etherized) 0.5 day-old males and females. No appreciable difference in migration or staining intensity could be detected between the bands for the frozen and those for the fresh flies, with respect to both isomerase and esterase.
Assays of glucose-6-phosphate dehydrogenaseand alkaline phosphatase These two enzymes were assayed by a direct measure of their activities, as a function of the age of the flies. Frozen flies were singly ground with a glass rod in 4 × 45 mm plastic centrifuge tubes containing 0.2 ml 0.01 M Tris, pH 7.2 + 0.005 M Mg ~+. The crude extracts were then centrifuged at 15,000 g for 1 min in a Beckman/Spinco Microfuge. The supernatant was assayed for G6PD activity after the method of DeMoss (1955). The reaction mixture consisted of (0.425 ml 0.02 M Tris, pH 7.4 + 0.002 M Mg 2+) + 0.025 ml 30 mg/ml glueose-6-phosphate disodium salt (Calbiochem) + 0.005 ml 7-65 mg/ml NADP + (Sigma) + 0.05 ml supernatant. The reduction of NADP + was measured as AOD/minute at 340 m~ with a Beckman DU Spectrophotometer and Gilford 2000 recorder. 6-phospho-gluconate dehydrogenase, the enzyme following G6PD in the hexose-mono-phosphate pathway, is also NADP + linked. This enzyme is very likely present in the extracts and, since the z~OD was recorded for at least 10 min, the AOD value really reflects the activity of the two enzymes. However, this does not in any way alter the purpose of the investigation; and, for the sake of simplicity, this activity will continue to be referred to as G6PD activity. In a preliminary experiment, the assay used here was found to be valid, i.e. linear with respect to concentration of the extract and with respect to time of reaction. Alkaline phosphatase was assayed after the procedure of Echols, Garen, Garen, and Torriani (1961). The reaction mixture contained 0.2 ml 1 mg/ml p-nitrophenyl phosphate di-sodium (NPP) (Mann Research Laboratories, New York, New York) in 1.0 M
J. c. HALL
210
Tris, p H 8.0 + 0.05 ml supernatant. T h e cleavage of orthophosphate from N P P was measured as A O D / m i n u t e at 410 m~t. Again, the assay proved valid. RESULTS T h e survival curves indicate that females who remain virgin throughout their lifetimes show greater survival than both females who mate, and males who mate or do not (Fig. 1 ; T a b l e 1). Males, on the other hand, yield virtually the same survival curve and mean lifespan whether they mate or not. As was expected, cultures which began with five pairs per vial yielded progeny for a far longer time than did the single pair mating cultures (Fig. 2).
F.
A Females only
~'~q
• B Moles only o [ Moles with females , • CIFemales with males I
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49
58
67
Days
Fic. 1. Survival curves for Drosophila melanogaster. Arrows represent times when 45-55 flies in experiments A and B were frozen for the enzyme assays. A: females only; 240 vials @ 10 females/vial. Day %surviving Day %surviving 1 100"00 46 91-76 4 99-54 49 88'58 7 99"25 52 83'13 10 99"04 55 75'99 13 98"91 58 64"23 16 98"74 61 48"07 19 98"58 64 27.39 22 98"37 67 12"16 25 98"11 70 5.13 28 97"85 73 2.30 31 97"17 76 0"91 34 96"35 79 0"16 37 95"84 82 0"05 40 95"02 85 0"00 43 93"69
AGE-DEPENDENTENZYME CHANGESIN DROSOPHILA MELANOGASTER
211
Fro. 1. continued
Day 1
4 7 10 13 16 19 22 25 2g 31 34 37 40 Day 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
B: males only; 240 vials @ 10 males/vial. % surviving Day 100.00 43 99"67 46 99.29 49 99.00 52 98.49 55 98.33 58 97"41 61 96.90 64 96.49 67 95.73 70 93.93 73 92.31 76 88.85 79 85.17 82
%surviving 79"96 72.65 63.85 56"40 43"60 34.44 24"71 15"90 94.8 4-43 1.59 0.63 0"06 0"00
C: females and males together; 240 vials @ 10 pain/vial. % females % males % females surviving surviving Day surviving 100"00 100"00 49 73"59 99"33 99"17 52 64"80 98"83 98"67 55 56"09 97"83 98"33 58 45"07 97"17 97"58 61 33"21 96"92 96"75 64 22-68 96"15 96"23 67 11"67 95"31 95"31 70 5"26 93"56 93"97 73 1'82 92-22 92"99 76 0"48 90"77 91'62 79 0"10 88"72 88"38 82 0"00 -86"84 86"41 85 84.44 81"37 88 -81.54 76.67 91 -77.44 71.37
% males surviving 64"96 56"00 46"22 37"78 29'67 22-20 12"25 7.94 5"74 3"25 1"15 0"38 0"19 0.10 0'00
TABLE 1. MEANLIFE SPANSOF Drosophila melanogaster Mean life span (days) Females only (10/vial) Males only (10/vial) Females with males (5 pairs/vial) Males with females (5 pairs/vial) Female with male (1 pair/vial) Male with female (1 pair/vial)
59.0 52.0 53.8 52.9 41.9 50.4
Figures 3a and b show the banding patterns for hexose-P-isomerase activity, as a function of age, for representative extracts. T h e r e was a high degree of similarity in migrating behavior and staining intensity of extracts within the various age groups. I n addition, the normalized distances migrated toward the anode by the bands were very nearly the s a m e - - b o t h within an age group and when comparing one age group to another.
212
j. C. HALL
I
• 5 females + 5 males/vial:
cultures
L ., ,21:r::e ; Yalf:;vm:inulful;s.2 days yield progeny for
mean of 2:5.5 days
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FIG. 2. Decline in fecundity of vial cultures with age.
Day 0-30 31 34 37 40 43 46 49
5 females + 5 males per vial: %culturesfecund Day 100.00 52 99"08 55 98.62 58 97.25 61 95.87 64 93.58 67 90.83 70 81"19
%culturesfecund 73"39 51.38 33.94 18"80 5-50 1.38 0-00
Day 1 4 7 10 13 16 19 22 25 28
1female + l male per vial: %culturesfeeund Day 98'32 31 98'32 34 95"80 37 94"96 40 89.08 43 80-67 46 72"27 49 57"14 52 40"34 55 23"53
%culturesfeeund 10"08 4"20 2"52 2"52 2'52 2"52 1"68 0'84 0"00
T h e changes noted in isomerase activity are (1) an increase in staining intensity for both females and males, first noticeable for 49 day-old flies and (2) a virtual disappearance of the two most rapidly m o v i n g (most anodal) bands for extracts f r o m 58 and 67 day-old females. An attempt to quantify the first type of change was m a d e as follows: Single-fly extracts were prepared in the same m a n n e r as described previously for the quantitative assays of G 6 P D and alkaline phosphatase. Change in ODs40 vs. time was
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A
MELANOGASTER
213
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Age, doys Origin FIG. 3. A g e - d e p e n d e n t changes in electrophoretic banding pattern of female hexose-P-isomerase
(3a) and male hexose-P-isomerase (3b). q- represents anode. For 0.5 day-old flies, 54 individuals were assayed; for each successive age group, 16 individuals were assayed. Each diagram is a representative result of these single fly assays. Aetual distances migrated by the bands ranged from c a . 2 to 4 crn from gel to gel. determined for the following reaction mixture: 1.0 ml 0-3 M Tris, pH 8.0 ÷ 0.1 ml 0.1 M Mg ~+ + 0.050 ml 10 mg/ml fructose-6-phosphate + 0.050 ml 6 mg/ml NADP + + 0.050 ml 1 U/ml G6PD + 0.050 ml extract supernatant. The assay proved valid; it revealed (Table 2) that 58 day-old females have 1.3 times more isomerase activity than 0.5 day-old females, and that 49 day-old males show a 1.5 fold increase over those 0.5 days old. The coefficients of variation C (standard deviation/ mean) for each age group assayed are very small confirming the visual observation of homogeneity of staining intensity on the gels. The amount of isomerase in extracts
214
J. c. HALL from old females was found to be greater than the amount in old males (Table 2); this, too, confirms the visual observation of very intensely stained isomerase bands for females 58 and 67 days old (Fig. 3a). TABLE 2. INCREASE IN HEXOSE-P-ISOMERASE ACTIVITY WITH AGE
Mean AODa40/min x 10 -4 4- standard deviation s: coefficient of variation C (no. flies assayed) Age (days) Females Males
0" 5 49 58 782 4- 65 : 0"08 -1005 ± 70 : 0.07 (12) (12) 577 4- 82:0"14 844 ~: 106:0"13 -(12) (12)
P (t-test)
< 0"005 < 0"005
T h e second type of isomerase c h a n g e - - a n alteration in banding pattern with a g e - was easily recognized since extracts from 0"5 day-old females were run in the same gels as the 58 and 67 day-old extracts. T h e fourth and fifth bands for the 0.5 day-old flies were fainter and narrower than were the first three bands (Fig. 3), but they were clearly delineable. Figures 4a and b show representative banding patterns for esterase activity. Again, there was much homogeneity within the various age groups; and the distances migrated by the bands were nearly the same within and between age groups. T h e changes with age for this enzyme were very striking: (1) a slight but noticeable change in intensity of staining for the females, first noticeable for 49-day-old flies; (2) a marked increase in staining intensity of the bands for the males, first seen for 25 day-old flies; and (3) the appearance of new bands, both slower and faster migrating, for the older males. T h e actual increase in amount of esterase in the extracts from old flies was quantified as follows: Extracts of single flies were prepared as described earlier; a change vs. time in ODss0 was determined for a reaction mixture consisting of: (10 -4 g oc naphthylacetate in 0.005 ml H 2 0 + 0.005 ml acetone) q- 1.0 ml 0.2 M PO~, p H 6.8 q- 0.4 nag fast red T R salt Jr 0"050 ml extract. This assay was found to be valid. T h e results of assaying several old and young flies (Table 3) show that extracts of 58 day-old females have TABLE
3. INCREASE IN ESTERASE ACTIVITY W I T H A G E
Mean AOD350/min × 10-4 standard deviation s: coefficient of variation C (no. flies assayed) Age (days) Females Males
0' 5 140 ± 30:0'21 (12) 122 4- 20:0"16 (12)
49 -319 4- 64:0"20 (12)
58 215 4- 44:0"20 (12) --
P (t-test)
< 0"005 < 0'005
AGE-DEPENDENT ENZYME CHANGES IN
DROSOPHILA
MELANOG~qSTER
215
1 "5 times as much esterase activity as do 0-5 day-old females, and that 49 day-old males have 2.6 times as much activity as males 0.5 days old. The coefficients of variation again are fairly small, confirming the observed homogeneity of staining intensity within age groups. And males show a greater increase with age in amount of esterase than do females, as was assumed to be so from the gels: old males yield esterase bands which stain very intensely relative to young males (Fig. 4b); whereas bands for old females exhibit less of a relative increase in staining intensity (Fig. 4a).
m
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FIG. 4. Age-dependent changes in electrophoretic banding pattern of female esterase (4a) a n d m a l e esterase (4b). + r e p r e s e n t s anode. F o r 0"5 day-old females a n d males, 108 a n d 54 individuals, respectively, were assayed; for each successive age group, 16 i n d i v i d u a l s were assayed. E a c h d i a g r a m is a representative result of these single-fly assays. Actual distances migrated by the bands ranged from c a . 4 to 6 c m from gel to gel.
216
j.C.
HALL
The newly appearing esterase bands were (a) three relatively faint and slower migrating bands first evident for 25 day-old males and appearing in all cases (i.e. for all old males assayed) (Fig. 4b); (b) varying numbers of faint, very rapidly migrating (more anodal) bands appearing for seven out of the forty-eight (14-6 per cent) males assayed who were 49, 58, and 67 days old (Table 4). It was thought that, perhaps, the appearance TAm~. 4.
DISTANCES MIGRATED BY RAPIDLY MOVING ESTERASE BANDS FOR OLD MALES : c a .
7-9
cm vs.
NORMAL Ca. 4--6 c m . T H E S E ADDITIONAL, FAINT BANDS APPEARED FOR 7 OUT OF 48 m c r R A c ' r s FOR MALES 49, 58, AND 67 days OLD. DISTANCESMIGRATED ARE NORMALIZED W I T H RESPECT TO DISTANCE MIGRATED BY MOST PROMINENT BAND FOR A 0 " 5 d a y - O L D EXTRACT SET EQUAL TO 1 • 0 0
Case
Age (days)
No. rapid bands
Normalized distance migrated
1
49
1
1"89
2
49
2
3
49
4
1"48 1" 96 1"46 1 "87 1 "91 1 "96
4
49
2
5 6 7
58 58 67
1 1 2
1"46 1 "87 1" 98 2"12 2" 14 2"29
of new bands was for a trivial reason: since esterase stains faintly for the 0.5 day-old males (and there is relatively little activity present), the fact that new bands appear in the more darkly staining banding patterns for the old flies might be because a threshold concentration of enzyme is necessary for these new bands to stain, and that this threshold is not met for the young flies. In short, esterase might be present--with respect to a 0.5 day-old male--in the locations on the gel corresponding to the place where "new" bands appear for the old extracts, but there is simply not enough of it to give a staining reaction. A crude answer to whether or not this was so was obtained by concentrating ten 0.5 dayold males--grinding them in the same volume of buffer (0.1 ml) normally used for single flies--and running this extract on the same gel as single old-fly extracts prepared in the usual manner. The result, a sample of which is shown in Fig. 5, was that the primary band for the concentrated extract appeared to stain every bit as intensely as did the primary band for an old male (49 days old); but (i) only one of the 3 "new" cathodal bands stained for the concentrated extract (and this very faintly); and (ii) no "new" rapidly migrating bands were detectable regarding the concentrated young male extract. As a preliminary to the quantitative assays of G6PD and alkaline phosphatase activity, flies of the various ages were weighed to see if any increase or decrease in activity with age which might be found would be accountable (perhaps trivially) to increases or decreases in body weight. The results (Table 5) show that females increase in mean weight (ca. 12 per cent) from an age of 0.5 day to 25 days but remain fairly constant thereafter. This is very likely because of egg production by these virgin females; presumably, more
AGE-DEPENDENT
ENZYME CHANGES IN D R O S O P H I L A
m
MELANOGASTER
217
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49 Age,
days
!O flies/O-ImL
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Fxo. 5. Comparison o f esterase banding patterns o f a concentrated extract f r o m young males w i t h a standard extract f r o m an old male.
eggs than were present at eclosion had piled up in the abdomens of the older females; and in fact, many eggs were seen on grinding up the older females for the enzyme assays. The males show no increase in weight over their lifetime, and in fact are of a fairly constant mean weight at all ages--with the curious exception of a c a . 18 per cent drop (compared with the mean value for all the other age groups) in body weight exhibited by 49 day-old males. T A B L E 5.
M E A N WEIGHTS OF FROZEN FLIES. EACH VALUE IS MEAN W E I G H T (PER FLY) I N m g ARRIVED AT BY W E I G H I N G 2 0 FLIES TOGETHER
Age (days)
Females
Males
0.5 25 49 58 67
1"173 1"333 1.375 1.405 1.380
0-845 0.893 0.718 0.845 0.920
The assays of the two enzymes showed marked changes in activity as a function of age; and, again, the changes were different for the two sexes. G6PD activity (Fig. 6) for females rose almost threefold to a peak for 49 day-old flies, returning by day 67 to the initial (day 0.5) activity; though the standard deviations for most age groups are large, this rise in activity and subsequent fall is highly significant by t-tests (P < 0.005 for both the rise and the fall). Activity for this enzyme from males increased more than two fold, with a peak for 25 day-old flies, then sank to almost negligible activity for extracts from the oldest flies; again, both the rise and the fall are significant (P < 0.005). Alkaline
218
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FIG. 6. Age-dependent changes in G6PD activity: mean AODs4o/min × 10.4 4- standard error of mean (standard deviation/~/n). A, Females; O, Males. Age (days) 0.5 25 49 58 67
Females 12 12 34 15 11
+ ± ± ± ~
1.0 1.2 3"8 2.6 2.3
Males 9 ± 0-8 23 ± 2-3 11 4- 0.9 7 ~ 0.3 3 + 0"6
phosphatase activity (Fig. 7) increased significantly (almost two-fold, P < 0.005) for the females, with a plateau for days 25 and 49, before falling to the level of initial activity by day 67. For the males, there was a greater than two-fold rise in activity to a peak for flies 49 days old. This increase is significant (P < 0.005) but the subsequent decrease for days 58 and 67 (to a level nearly 60 per cent higher than that for the youngest males) is not quite significant (P < 0.10). It should be noted that even if the weight increase for females (youngest vs. all older age groups) is taken into account, the changes which are seen are still highly significant. DISCUSSION Both the electrophoretic and the quantitative enzyme assays reveal patterns of agedependent changes which are markedly different for the two sexes. Komma (1968) has shown that G6PD produced by young Drosophila males differs from that produced by young females in terms of electrophoretic migration, kinetics, and stability. Further, this difference persisted in mixed homogenates of males and females, implying that something more than the temporary binding of small molecules is involved. Thus there may be fundamentally different physiological conditions prevailing in males and females with respect to enzyme synthesis. Such differences might account for the very different
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A 4OO -
MELANOGASTER
219
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FIQ. 7. Age-dependent changes in alkaline phosphatase activity: mean AOD410/min × 10 -4 ± standard error of mean (standard deviation/~/n). A, Females; 0, Males. Age (days) 0"5 25 49 58 67
Females 169 335 318 228 175
i ± ~ ± ±
10"6 38"9 31"8 30-0 11"8
Males 107 116 219 180 164
~ ± i ± ±
9"6 8"4 40"4 25"7 28"0
N.B. For Fig. 6 and for Fig. 7, n = 24 flies for 0"5 day-old females and males; and n = 12 flies for females and males from all other age groups. Each point is a mean value :k one standard error of mean, resulting from 24 single-fly assays for 0" 5 day-old flies, and 12 such assays for flies from all other age groups. patterns of enzyme change with age that have been shown here. And these sex-specific enzyme changes might themselves be reflected in the different survival curves shown by males and females; that is, genetically influenced senescence might be mediated b y rather different processes in male and female Drosophila. A question arises as to whether the age-dependent peaks of enzyme activity result from m o r e enzyme being made or f r o m a more active enzyme being made in the same a m o u n t as for young individuals. Schneiderman, Young, and Childs (1966) feel that Drosophila alkaline phosphatase is a family of enzymes whose m e m b e r s are organ- or even tissue-specific. Perhaps a different, m o r e active alkaline phosphatase is made in flies 49 days old than is m a d e in newly emerged adults; and perhaps such a switch is intimately involved in senescence. T h e results reported here do point the way toward some questions as to how an organism's changing biochemical constitution might be
220
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related to the process of senescence. Butit is clear that one cannot use only these survival curves and the results of the several assays meaningfully to construct specific, detailed models on the causes and mechanisms of aging. The purpose of the investigation has, however, been fulfilled: some objective, fairly precise symptoms of aging fruit flies have been established; at this point is is not crucial to causally relate these enzyme changes to a precise mechanism of senescence. But there is now some basis for trying to find possible "aging mutants": genetic variants of D. melanogaster which show patterns of enzyme changes different from those established here. One could, for example, mutagenize chromosomes or collect them from nature, make them homozygous, and then ask if there was induced or if there existed in nature a mutation whose phenotype is--with respect to the wild-type changes now established-repeatably different changes in enzyme banding patterns (earlier or later changes or ones that are altogether different in kind) and different patterns of increases and decreases in enzyme activity.
Acknowledgements--I should like to thank Drs. L. SANDLER,J. GALLANT and A. MOTULSKY for their guidance, comments and interest. I am also indebted to Drs. N. CARTER, J. GALLANT,and J. IRR for their expert technical instruction.
REFERENCES CASAR~rr, G. W. (1964) Adv. gerontol. Res. 1, 109. CHAPMAN,R. G., Hm~NmSEY, M. A., WALTERSDORPH,A. M., HUP-~EKENS, F. M. and G~'u3to, B. W. (1962)j~. din. Invest. 41, 1249. DEMoss, R. D. (1955) In Methods in Enzymology, (Edited by COLOWICK, S. P. and KAPL~"L N. O.) Vol. 1, p. 328. Academic Press, New York. ECHOLS, H., G ~ N , S. and TORRIP~'~I, A. (1961) J. mol. Biol. 3, 425. EPSTEIN, C. J., MARTIN, G. M., SCHULTZ, A. L. and MOTULKSY, A. G. (1966) Medicine 45, 177. GONZALEZ, B. M. (1923) drner. Naturalist 57, 289. HARRIS, H., HOPKINSON, D. A. and ROBSON, E. B. (1962) Nature, Lond. 196, 1296. KOMMA, D. J. (1968) Biochem. Genetics 1, 337. ROCKSTmN, M. (1966) In Topics in the Biology of Aging (Edited by KROHN, P. L.) p. 28. Interscience Publishers, New York, London and Sydney. SCHNEIDERMAN,H., YOUNG, W. J. and CHILDS,B. (1966) Science 151, 461. SMITHIES, O. (1955) Biochem. J. 61, 625. SZlLARO, L. (1959) Proc. Natl..4cad. Sci. U.S. 45, 30. S u m m a r y - - 1 . Marked age-dependent changes in four enzymes were found by assaying crude extracts of Drosophila melanogaster. A. Hexose-P-isomerase from very old females lacked two of the five bands characteristic of young female extracts run on starch gels; the staining intensity increased for the older females and for older males. B. Esterase from old males showed several additional electrophoretic bands which did not appear in assays of young males; in addition there was a large increase in staining intensity for male esterase and a lesser such increase for females. C. G 6 P D activity--quantitatively determined---showed a significant increase and then a decrease as a function of age; peak activity of the male enzyme was for flies 25 days old, while this peak appeared at 49 days for females. D. Alkaline phosphatase activity showed similar significant increases and decreases with age, with a plateau of activity occurring at 25 and 49 days for females, and a peak appearing with respect to males 49 days old. 2. Survival curves for flies kept in shell vials were determined. T h e y show later mortality for virgin females than for females who mate. Mated females had a slightly longer lifespan than mated or unmated males.
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A
MELANOGASTER
3. These symptoms of aging Drosophila were established to form a basis for looking for, then recognizing, genetic variants whose phenotype is an altered pattern of senescent change. Characterization of such "aging mutants" could provide ways of understanding the process of senescence. R 6 a u m 6 - - 1 . Des alt6rations marqu6es, en fonction de l'age, de quatre enzymes ont 6t6 relev6es au titrage d'extraits crus de Drosophila melanogaster. A. L'hexose-P-isom6rase de tr~s vieilles femelles 6tait priv6e de deux des cinq bandes caract6ristiques des extraits de femelles jeunes port6s sur des gels d'amidon; l'intensit6 de coloration augmentait dans le cas des males et des femelles plus ag6s. B. L'est6rase de mMes ag6s pr6sentait ~ l'61ectrophor~se plusieurs bandes additionnelles, qui ne se manifestaient pas lors des titrages d'extraits de jeunes males; en outre, l'intensit6 de coloration augmentait fortement dans l'est6rase des males et beaucoup moins dans celle des femelles. C. D&ermin6e quantitativement, l'activit6 de la G6PD manifestait une augmentation significative, puis une diminution en fonction de l'age; le pic d'activit6 de l'enzyme se situait chez les mouches males ~ l'age de 25 jours, mais ne se manifestait qu'h 49 jours chez les femelles. D. L'activit6 de la phosphatase alcaline a manifest6 de semblables augmentations et diminutions significatives avec l'age, avec u n plateau d'activit6 A 25 et 49 jours pour les femelles et u n pic ~ 49 jours chez les males. 2. Les courbes de survie des mouches gard6es en flacons ont 6t6 d&ermin6es. Elles r6v~lent une mortalit6 plus tardive chez les femelles vierges que chez les femelles accoupl6es. La long6vit6 de ces derni~res &ait 16g6rement sup6rieure que celle des males, accoupl6s ou non. 3. Ces sympt6mes du vieillissement chez Drosophila ont 6t6 relev6s pour constituer une base d'examen, puis d'identification de viarantes g6n&iques dont le ph6notype est u n ensemble alt6r6 de modifications s6nescentes. La caract6risation de ces "mutants vieillissants" pourrait ouvrir des voles h la compr6hension du processus de s6nescence. Z u s a m m e n f a s s u n g - - 1 . I n Rohextrakten yon Drosophila melanogaster wurden ausgepr~igte altersabhiingige Ver~inderungen bei vier Enzymen gefunden. A. Bei Hexosephosphatisomerase von sehr alten Weibchen fehlten in Stiirkegelliiufen zwei der fiinf Banden, welche charakteristisch fiJr junge Weibchen sind. Die Anfiirbbarkeit stieg bei den iilteren Weibchen und Miinnchen. B. Esterase alter M~innchen zeigte mehrere zusiitzliche elektrophoretische Banden, die bei jungen Miinnchen nicht zu finden waren. AuBerdemstieg die Anffirbbarkeit bei Esterase von Miinnchen stark an, weniger stark bei Weibchen. C. Die quantitativ bestimmte G6PDH-Aktivitiit zeigt in Abhangigkeit vom Alter zun~ichst einen deutlichen Anstieg, dann einen Abfall. Der Gipfel lag bei m~nnlichen Fliegen beim 25. Lebenstag, bei Weibchen bei 49 Tagen. D. Die Aktivitiit der alkalischen Phosphatase zeigte iihnliche altersabhiingige Anstiege und Abnahmen. Ein Plateau trat bei Weibchen bei 25 u n d 49 Tagen auf. Bei Miinnehen lag der Gipfel am 49. Lebenstag. 2. Fiir in Schalenfliischen gehaltene Fliegen wurden die l~bedebenskurven bestimmt. Fiir unbefruchtete Weibchen ist die Lebensdauer grOBer als f'dr befruchtete. Befruchtete Weibchen hatten etwas l~ngere Lebensdauer als M~mchen. Die gilt gleichermaBen fiir gepaart habende u n d fiir nicht gepart habende Miinnchen. 3. Diese Befunde bei alternden Drosophila sollen eine Basis fiir die Suche u n d Erkennung yon genetischen Varianten mit ver~_nderten Phiinotyp der Altersveriinderungen bieten. Die Charakterisierung solcher "Alternsmutanten" k6nnte zum Verst~ndnis der Altersprozesse beitragen.
221
222
j . c . HALL P e ~ m M e - - 1 . [ I p H a u a J m 3 e Heo6pa6oTammLX 3KcTpaKTOB H3 Drosophila melanogaster 6 b L ~ O6Hapy'~eHH 3aMeTH~e H3MeHeHI.DI, 3aBHcsn~He OT Bo3pacTa, B qeT~pCx [~pMeHTaX. A . B r c K c o 3 e - - - P - - H 3 0 M e p a 3 e H 3 0 q e a b cTapI~x CaMOK a e ~ o c T a B a n o ~I~yx H3 I]~qTH IIOJIO¢, x a p a x T ~ p l ~ i X ~lJI~l3KCTpaKTOB 143 MOJIO]IbIX caMoK IIpH HCIIMTaHHH Ha KpaxMaJibm i x re~-~x; y 6oHee cTapbIx CaMOK H CaMlleB y s e m o m ~ a c s HHTeHCHBHOCTb oKpacKH. B. ~ T e p a 3 a H3 cTapsIX CaMIIOB BbLqBHHa HOCKOHbKO ROHOJIHHTenbHbI X3HeKTpo~bopeTHqeCKHX IIOJIOC, KOTOpMe He O6Hapy3KHBaJIHCI, npH aHa~H3e y MO~O~bLX CaMUOS; KpOM¢ TOrO, 6ht~O 6o~IbmOe noBbimeHHe B HHTeHCTIBHOCTH oKpacKH 3cTepa3bx CaMUOB H M~H~ B~ipamem~oe n o e b l m e t m e y caMoK. B. AKT~SHOCTb r m o x o 3 b x 6-~boc0aTa ~lerm~poreHa3m ( G 6 P D ) - - n p H ~Om~e~rBeHHOM o n p e ~ e a e m m m, m s s n a c H a q a ~ a 3Ha~mTemsHoe n o s b ~ m e t m e , a 3aTOM CHH~eHHe C s o z p a c TOM; BbICl~a.q T O g a aKTHBHOCTH ~pepMeHTa CaMHOB o 6 H a p y m t m a n a c b y Myx B e o 3 p a ~ r e 25-H ~Hett, T o r ~ a Ka~ y caMoK rm~ HO~BJLqHC~ ~ Bo3pacTe 49-H ~Hett. ~ . Axw~m~OCWb m e n o ~ m o a ~boc~bawa3bi o 6 H a p y m H s a ~ a CXO~HSIe 3Ha~HTeJIbSble I~OBbImeHmq H CHH~¢HH~ C eo3pacTOM, npH~¢M BbICHIH~ y p o e e m , aKTHBHOCTH y CaMO~ IlOCTHraJICll B Bo3pacwe 25--rt H 49--H ~I8el:i, a y caMraoa a Bo3pacTe 4 9 - r t ./~Hel~. 2. Onpe/Iemumc~, xprem],ie m,~acnaaeMocTrl ~ n a Myx rlpH co~lep~armH B paKOBHHH],~X c ~ q n K a x . O n n o6Hapyr, o t r m 6 o n c e noa,aHrOIO CMepTHOC'rb y ,KeBCTBOHHblX CaMOK, qeM y c n a p H a a r o m H x c ~ CaMOK. ~ cnapeHHMX CaMOK ~'IHT~JIbHOCTb ~EH3HH 6 u n a HeCKOJIbI(O BbIme, qeM y cnapenHbLX H HecHapeHH/~IX CaMI~OB. 3. ~TH CHMIITO1VEbl cTapeHHI/ y Drosophi[~l ~bIfIH yCTaHOB~IeHI~I KaK OCHOBa~ HCXO~$I H3 KOTOpOI~ MO3KHO HCKaTb~ a 3aTeM pacIIO3HaTb, rCHeTI~eCKHe BapHaHTbI, (I)eHOTHH KOTOpbIX npe~CTaBJDIeT CO~Oi~ BH~OH3MCHeHHe THHa H3MeHeHH~ HpH CTapeHHH. XapaKTepH3al~H~l TaKHX "CTapetoI/~HX MyTaHTOB" MO~I£eT BbIIIBHTb ~aHHble ~rLq HOHHMaHH~I H p o u e c c a cTapCHH~I.
A G E - D E P E N D E N T ENZYME CHANGES IN DROSOPHILA MELANOGASTER* J. C. HALL Department of Genetics, University of Washington, Seattle, Washington 98105
(Received 29 May 1969)
INTRODUCTION THE PURPOSEof this investigation is to seek ways of examining the involvement of the genome of Drosophila melanogaster in the process of senescence. Genetic causes of aging may be of two kinds: (1) built-in errors or contradictions of design (faults of the genotype as established at the time of fertilization); and (2) somatic mutations (microaccidents which disrupt the structure of genes or chromosomes). Evidence bearing on somatic mutation theories of aging (such as that of Szilard, 1959) is in some cases consistent, but in others so contradictory that one can say, at least, that it is improbable that aging in general is a consequence of the accumulation with time of somatic gene mutations or chromosome aberrations. To examine the first possibility--a genetic determination of senescence--it would be desirable to search for, and characterize, mutations that affect aging. Gonzalez (1923) demonstrated, in D. melanogaster, that abnormalities in structure caused by the presence of homozygous mutant genes (such as purple eye color) cause a decrease in longevity. But a study of such mutants would say little about senescence, for the only connection they have with aging is that they lead to early death. The kind of inherited faults which could be termed true aging mutations would be genetic lesions whose phenotype is a mimicry of aging based on a definite set of criteria of the phenomenon in addition to decreased lifespan. Casarett (1964) has put forth criteria which can be used to detect a mutation whose phenotype is premature aging. A population of individuals carrying this putative aging mutation would show (1) an earlier increase in mortality without alteration of the shape of the mortality curve; (2) a proportional advancement in time of all diseases or causes of death; (3) a proportional advancement in time of all morphological and physiological manifestations of the aging process. There are a few mutations known which seem to result in a form of premature senescence. Werner's syndrome in man is inherited as a single autosomal recessive mutation (Epstein, Martin, Schultz and Motulsky, 1966). Many (though not all) of the symptoms of this condition, which first appear at about age twenty in individuals homozygous for the lesion, are quite similar to features manifested in normal human senescence. But Epstein et al. (1966) are very cautious as to whether this disease is truly precocious or accelerated senescence, or whether it may better be considered a "caricature" of aging. * Research sponsored jointly by Grant 5 T01 GM 00182 and Grant RG-9965, both from the U.S. Public Health Service. 207
208
j.C. H&LL
In any event, man is in many ways a refractory organism with which to study the the nature of a genetic alteration. Drosophila melanogaster, on the other hand, has a powerful genetic system; the advantage that many individuals can be screened for a putative aging mutation; and the property that survival curves for flies carrying the putative mutation can easily be determined with enough organisms to make the shapes of the curves meaningful. However, very little is known of the diseases or causes of death in normal Drosophila, or of the morphological and physiological features of aging. The purpose of this study, then, is to determine some of the symptoms of senescence in fruit flies; that is, to describe changes which can be consistently found to occur over the life of the organism. Against such a background, a search for inherited changes which alter the aging process could be carried out. Rockstein (1966) has found that there are changes in the activities of certain enzymes which can be demonstrated in aging male house flies (Musca domestica L). The plan of the present investigation is to ask whether enzyme changes---of a qualitative and quantitative nature---occur in aging Drosophila melanogaster. It has been found that of four enzymes examined (two by electrophoresis, two by a direct measure of activity), all show changes as a function of the age of the flies from which they were extracted. These enzymes, moreover, were not chosen because of any a priori suspicion that they were especially related to senescence. METHODS AND PROCEDURE
Survival curves The Drosophila melanogaster stock used in these experiments was a Canton-S wild type. Virgin females and males whose age varied from 0 to 12 hours were collected from bottle cultures. Four separate experiments were begun at the time of collection: (A) ten females per vial (240 vials), (B) ten males per vial (240 vials), (C) five females and five males per vial (240 vials), and (D) one female and one male per vial (120 vials). Also at this time a large number of excess females and males were quick-frozen in an ethanol plus dry ice bath and stored at - 60°C. At 72-hr intervals, the flies in the four experiments were transferred to fresh vials, with the number of survivors being noted at each transfer. In addition, the old cultures in experiments (C) and (D) were saved so that they could be scored later for the presence or absence of progeny. On the 25th, the 49th, the 58th, and the 67th day from the beginning of the experiments, 45-55 flies from experiments (A) and (B) were etherized, quick-frozen, and stored as before, to be used for the enzyme assays.
Assays of hexose-P-isomerase and esterase These two enzymes were assayed by starch gel electrophoresis to see if an altered banding pattern would appear as a function of the age oftheflies. Frozen flies were singly ground with a glass rod in 10 × 75 mm glass test tubes containing 0.1 ml 0.05 M PO~ buffer, pH 6.5. Starch gel electrophoresis was done according to the method of Smithies (1955). Each gel was made with 44.0 g starch (Electrostarch Company, Madison, Wisconsin) in 400 ml 0"005 M PO4, pH 7"0. The buffer in the gel boxes was 0.1 M PO~, pH 7-0. Paper inserts (a double thickness of Whatman No. 3 filter paper) soaked in the crude fly extracts were placed in the gel, and a potential difference of 10.2 V/cm was applied across the length of the gel for 4 hr. To normalize the distances traveled by the
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A M E L A N O G A S T E R
209
enzymes from gel to gel, an insert soaked in "reference extract" (15 0.5 day-old males or females ground in 1.5 ml buffer) was placed in each gel alongside the experimental inserts. When assaying extracts of the old flies, two of the twenty inserts in each gel were soaked in extracts from the 0.5 day-old flies; a direct comparison of migration and staining intensity of the enzymes from old flies vs. those from young flies was thus possible. At the conclusion of a run, the gel was sliced in half and the top half was stained by the method of Chapman, Hennessey, Waltersdorph, Huennekens, and Garbio (1962) for hexose-P-isomerase activity. The reaction mixture was modified to be as follows: 5-0 ml 0.3 M Tris buffer, pH 8.0 + 0.5 ml 0.1 M Mg 9"+ + 0.2 ml 10 mg/ml D-fructose-6phosphate disodium salt (Sigma Chemical Company, St. Louis, Missouri) + 0.2 ml 6 mg/ml NADP + (Sigma) + 0.3 ml 1 U/ml glucose-6-phosphate dehydrogenase (Calbiochem, Los Angeles, California) + 1 mg phenazine methosulfate (Sigma) + 1 mg M T T tetrazolium (Sigma) + 10 per cent agar (dissolved in hot distilled water then cooled to less than 45°C). The bottom half of the gel was stained after the method of Harris, Hopkinson, and Robson (1962) for esterase activity. The reaction mixture consisted of (0.02 g oc naphthylacetate (Sigma) in 1 ml H20 + 1 ml acetone) + 100 ml 0-2 M PO~, pH 6.8 + 40 mg fast red TR salt (Sigma). As a preliminary experiment, a control gel was run in which were inserted extracts from frozen 0.5 day-old males and females, and extracts from fresh (just-etherized) 0.5 day-old males and females. No appreciable difference in migration or staining intensity could be detected between the bands for the frozen and those for the fresh flies, with respect to both isomerase and esterase.
Assays of glucose-6-phosphate dehydrogenaseand alkaline phosphatase These two enzymes were assayed by a direct measure of their activities, as a function of the age of the flies. Frozen flies were singly ground with a glass rod in 4 × 45 mm plastic centrifuge tubes containing 0.2 ml 0.01 M Tris, pH 7.2 + 0.005 M Mg ~+. The crude extracts were then centrifuged at 15,000 g for 1 min in a Beckman/Spinco Microfuge. The supernatant was assayed for G6PD activity after the method of DeMoss (1955). The reaction mixture consisted of (0.425 ml 0.02 M Tris, pH 7.4 + 0.002 M Mg 2+) + 0.025 ml 30 mg/ml glueose-6-phosphate disodium salt (Calbiochem) + 0.005 ml 7-65 mg/ml NADP + (Sigma) + 0.05 ml supernatant. The reduction of NADP + was measured as AOD/minute at 340 m~ with a Beckman DU Spectrophotometer and Gilford 2000 recorder. 6-phospho-gluconate dehydrogenase, the enzyme following G6PD in the hexose-mono-phosphate pathway, is also NADP + linked. This enzyme is very likely present in the extracts and, since the z~OD was recorded for at least 10 min, the AOD value really reflects the activity of the two enzymes. However, this does not in any way alter the purpose of the investigation; and, for the sake of simplicity, this activity will continue to be referred to as G6PD activity. In a preliminary experiment, the assay used here was found to be valid, i.e. linear with respect to concentration of the extract and with respect to time of reaction. Alkaline phosphatase was assayed after the procedure of Echols, Garen, Garen, and Torriani (1961). The reaction mixture contained 0.2 ml 1 mg/ml p-nitrophenyl phosphate di-sodium (NPP) (Mann Research Laboratories, New York, New York) in 1.0 M
J. c. HALL
210
Tris, p H 8.0 + 0.05 ml supernatant. T h e cleavage of orthophosphate from N P P was measured as A O D / m i n u t e at 410 m~t. Again, the assay proved valid. RESULTS T h e survival curves indicate that females who remain virgin throughout their lifetimes show greater survival than both females who mate, and males who mate or do not (Fig. 1 ; T a b l e 1). Males, on the other hand, yield virtually the same survival curve and mean lifespan whether they mate or not. As was expected, cultures which began with five pairs per vial yielded progeny for a far longer time than did the single pair mating cultures (Fig. 2).
F.
A Females only
~'~q
• B Moles only o [ Moles with females , • CIFemales with males I
I
l
I
25
49
58
67
Days
Fic. 1. Survival curves for Drosophila melanogaster. Arrows represent times when 45-55 flies in experiments A and B were frozen for the enzyme assays. A: females only; 240 vials @ 10 females/vial. Day %surviving Day %surviving 1 100"00 46 91-76 4 99-54 49 88'58 7 99"25 52 83'13 10 99"04 55 75'99 13 98"91 58 64"23 16 98"74 61 48"07 19 98"58 64 27.39 22 98"37 67 12"16 25 98"11 70 5.13 28 97"85 73 2.30 31 97"17 76 0"91 34 96"35 79 0"16 37 95"84 82 0"05 40 95"02 85 0"00 43 93"69
AGE-DEPENDENTENZYME CHANGESIN DROSOPHILA MELANOGASTER
211
Fro. 1. continued
Day 1
4 7 10 13 16 19 22 25 2g 31 34 37 40 Day 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
B: males only; 240 vials @ 10 males/vial. % surviving Day 100.00 43 99"67 46 99.29 49 99.00 52 98.49 55 98.33 58 97"41 61 96.90 64 96.49 67 95.73 70 93.93 73 92.31 76 88.85 79 85.17 82
%surviving 79"96 72.65 63.85 56"40 43"60 34.44 24"71 15"90 94.8 4-43 1.59 0.63 0"06 0"00
C: females and males together; 240 vials @ 10 pain/vial. % females % males % females surviving surviving Day surviving 100"00 100"00 49 73"59 99"33 99"17 52 64"80 98"83 98"67 55 56"09 97"83 98"33 58 45"07 97"17 97"58 61 33"21 96"92 96"75 64 22-68 96"15 96"23 67 11"67 95"31 95"31 70 5"26 93"56 93"97 73 1'82 92-22 92"99 76 0"48 90"77 91'62 79 0"10 88"72 88"38 82 0"00 -86"84 86"41 85 84.44 81"37 88 -81.54 76.67 91 -77.44 71.37
% males surviving 64"96 56"00 46"22 37"78 29'67 22-20 12"25 7.94 5"74 3"25 1"15 0"38 0"19 0.10 0'00
TABLE 1. MEANLIFE SPANSOF Drosophila melanogaster Mean life span (days) Females only (10/vial) Males only (10/vial) Females with males (5 pairs/vial) Males with females (5 pairs/vial) Female with male (1 pair/vial) Male with female (1 pair/vial)
59.0 52.0 53.8 52.9 41.9 50.4
Figures 3a and b show the banding patterns for hexose-P-isomerase activity, as a function of age, for representative extracts. T h e r e was a high degree of similarity in migrating behavior and staining intensity of extracts within the various age groups. I n addition, the normalized distances migrated toward the anode by the bands were very nearly the s a m e - - b o t h within an age group and when comparing one age group to another.
212
j. C. HALL
I
• 5 females + 5 males/vial:
cultures
L ., ,21:r::e ; Yalf:;vm:inulful;s.2 days yield progeny for
mean of 2:5.5 days
I 0 0 I-4--4--4--A,-~-- •-- •-- • - - 4 - - A ~ • _ • .
\e
%•
\ J
\
50
\
\ \
\
\
\
\ \
%
I
2O
40
\
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Days
FIG. 2. Decline in fecundity of vial cultures with age.
Day 0-30 31 34 37 40 43 46 49
5 females + 5 males per vial: %culturesfecund Day 100.00 52 99"08 55 98.62 58 97.25 61 95.87 64 93.58 67 90.83 70 81"19
%culturesfecund 73"39 51.38 33.94 18"80 5-50 1.38 0-00
Day 1 4 7 10 13 16 19 22 25 28
1female + l male per vial: %culturesfeeund Day 98'32 31 98'32 34 95"80 37 94"96 40 89.08 43 80-67 46 72"27 49 57"14 52 40"34 55 23"53
%culturesfeeund 10"08 4"20 2"52 2"52 2'52 2"52 1"68 0'84 0"00
T h e changes noted in isomerase activity are (1) an increase in staining intensity for both females and males, first noticeable for 49 day-old flies and (2) a virtual disappearance of the two most rapidly m o v i n g (most anodal) bands for extracts f r o m 58 and 67 day-old females. An attempt to quantify the first type of change was m a d e as follows: Single-fly extracts were prepared in the same m a n n e r as described previously for the quantitative assays of G 6 P D and alkaline phosphatase. Change in ODs40 vs. time was
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A
MELANOGASTER
213
÷ m n m
~m
m
m
m
n m
i..!°) 0"5I
r 25
I 49 5B Age, doy$ Origin
I
I
67
÷ m m i
m
m
m
m
m
N
(b) 05
I
I
25
49
I
58
I
67
Age, doys Origin FIG. 3. A g e - d e p e n d e n t changes in electrophoretic banding pattern of female hexose-P-isomerase
(3a) and male hexose-P-isomerase (3b). q- represents anode. For 0.5 day-old flies, 54 individuals were assayed; for each successive age group, 16 individuals were assayed. Each diagram is a representative result of these single fly assays. Aetual distances migrated by the bands ranged from c a . 2 to 4 crn from gel to gel. determined for the following reaction mixture: 1.0 ml 0-3 M Tris, pH 8.0 ÷ 0.1 ml 0.1 M Mg ~+ + 0.050 ml 10 mg/ml fructose-6-phosphate + 0.050 ml 6 mg/ml NADP + + 0.050 ml 1 U/ml G6PD + 0.050 ml extract supernatant. The assay proved valid; it revealed (Table 2) that 58 day-old females have 1.3 times more isomerase activity than 0.5 day-old females, and that 49 day-old males show a 1.5 fold increase over those 0.5 days old. The coefficients of variation C (standard deviation/ mean) for each age group assayed are very small confirming the visual observation of homogeneity of staining intensity on the gels. The amount of isomerase in extracts
214
J. c. HALL from old females was found to be greater than the amount in old males (Table 2); this, too, confirms the visual observation of very intensely stained isomerase bands for females 58 and 67 days old (Fig. 3a). TABLE 2. INCREASE IN HEXOSE-P-ISOMERASE ACTIVITY WITH AGE
Mean AODa40/min x 10 -4 4- standard deviation s: coefficient of variation C (no. flies assayed) Age (days) Females Males
0" 5 49 58 782 4- 65 : 0"08 -1005 ± 70 : 0.07 (12) (12) 577 4- 82:0"14 844 ~: 106:0"13 -(12) (12)
P (t-test)
< 0"005 < 0"005
T h e second type of isomerase c h a n g e - - a n alteration in banding pattern with a g e - was easily recognized since extracts from 0"5 day-old females were run in the same gels as the 58 and 67 day-old extracts. T h e fourth and fifth bands for the 0.5 day-old flies were fainter and narrower than were the first three bands (Fig. 3), but they were clearly delineable. Figures 4a and b show representative banding patterns for esterase activity. Again, there was much homogeneity within the various age groups; and the distances migrated by the bands were nearly the same within and between age groups. T h e changes with age for this enzyme were very striking: (1) a slight but noticeable change in intensity of staining for the females, first noticeable for 49-day-old flies; (2) a marked increase in staining intensity of the bands for the males, first seen for 25 day-old flies; and (3) the appearance of new bands, both slower and faster migrating, for the older males. T h e actual increase in amount of esterase in the extracts from old flies was quantified as follows: Extracts of single flies were prepared as described earlier; a change vs. time in ODss0 was determined for a reaction mixture consisting of: (10 -4 g oc naphthylacetate in 0.005 ml H 2 0 + 0.005 ml acetone) q- 1.0 ml 0.2 M PO~, p H 6.8 q- 0.4 nag fast red T R salt Jr 0"050 ml extract. This assay was found to be valid. T h e results of assaying several old and young flies (Table 3) show that extracts of 58 day-old females have TABLE
3. INCREASE IN ESTERASE ACTIVITY W I T H A G E
Mean AOD350/min × 10-4 standard deviation s: coefficient of variation C (no. flies assayed) Age (days) Females Males
0' 5 140 ± 30:0'21 (12) 122 4- 20:0"16 (12)
49 -319 4- 64:0"20 (12)
58 215 4- 44:0"20 (12) --
P (t-test)
< 0"005 < 0'005
AGE-DEPENDENT ENZYME CHANGES IN
DROSOPHILA
MELANOG~qSTER
215
1 "5 times as much esterase activity as do 0-5 day-old females, and that 49 day-old males have 2.6 times as much activity as males 0.5 days old. The coefficients of variation again are fairly small, confirming the observed homogeneity of staining intensity within age groups. And males show a greater increase with age in amount of esterase than do females, as was assumed to be so from the gels: old males yield esterase bands which stain very intensely relative to young males (Fig. 4b); whereas bands for old females exhibit less of a relative increase in staining intensity (Fig. 4a).
m
m
k
(a) ] 0.5
P 25
l 49
Age,
I 58
r 67
I 58
I 67
days
Origin
÷ m
i
(b) k 0 5
] 25
I 49 Age,
doys
Origin
FIG. 4. Age-dependent changes in electrophoretic banding pattern of female esterase (4a) a n d m a l e esterase (4b). + r e p r e s e n t s anode. F o r 0"5 day-old females a n d males, 108 a n d 54 individuals, respectively, were assayed; for each successive age group, 16 i n d i v i d u a l s were assayed. E a c h d i a g r a m is a representative result of these single-fly assays. Actual distances migrated by the bands ranged from c a . 4 to 6 c m from gel to gel.
216
j.C.
HALL
The newly appearing esterase bands were (a) three relatively faint and slower migrating bands first evident for 25 day-old males and appearing in all cases (i.e. for all old males assayed) (Fig. 4b); (b) varying numbers of faint, very rapidly migrating (more anodal) bands appearing for seven out of the forty-eight (14-6 per cent) males assayed who were 49, 58, and 67 days old (Table 4). It was thought that, perhaps, the appearance TAm~. 4.
DISTANCES MIGRATED BY RAPIDLY MOVING ESTERASE BANDS FOR OLD MALES : c a .
7-9
cm vs.
NORMAL Ca. 4--6 c m . T H E S E ADDITIONAL, FAINT BANDS APPEARED FOR 7 OUT OF 48 m c r R A c ' r s FOR MALES 49, 58, AND 67 days OLD. DISTANCESMIGRATED ARE NORMALIZED W I T H RESPECT TO DISTANCE MIGRATED BY MOST PROMINENT BAND FOR A 0 " 5 d a y - O L D EXTRACT SET EQUAL TO 1 • 0 0
Case
Age (days)
No. rapid bands
Normalized distance migrated
1
49
1
1"89
2
49
2
3
49
4
1"48 1" 96 1"46 1 "87 1 "91 1 "96
4
49
2
5 6 7
58 58 67
1 1 2
1"46 1 "87 1" 98 2"12 2" 14 2"29
of new bands was for a trivial reason: since esterase stains faintly for the 0.5 day-old males (and there is relatively little activity present), the fact that new bands appear in the more darkly staining banding patterns for the old flies might be because a threshold concentration of enzyme is necessary for these new bands to stain, and that this threshold is not met for the young flies. In short, esterase might be present--with respect to a 0.5 day-old male--in the locations on the gel corresponding to the place where "new" bands appear for the old extracts, but there is simply not enough of it to give a staining reaction. A crude answer to whether or not this was so was obtained by concentrating ten 0.5 dayold males--grinding them in the same volume of buffer (0.1 ml) normally used for single flies--and running this extract on the same gel as single old-fly extracts prepared in the usual manner. The result, a sample of which is shown in Fig. 5, was that the primary band for the concentrated extract appeared to stain every bit as intensely as did the primary band for an old male (49 days old); but (i) only one of the 3 "new" cathodal bands stained for the concentrated extract (and this very faintly); and (ii) no "new" rapidly migrating bands were detectable regarding the concentrated young male extract. As a preliminary to the quantitative assays of G6PD and alkaline phosphatase activity, flies of the various ages were weighed to see if any increase or decrease in activity with age which might be found would be accountable (perhaps trivially) to increases or decreases in body weight. The results (Table 5) show that females increase in mean weight (ca. 12 per cent) from an age of 0.5 day to 25 days but remain fairly constant thereafter. This is very likely because of egg production by these virgin females; presumably, more
AGE-DEPENDENT
ENZYME CHANGES IN D R O S O P H I L A
m
MELANOGASTER
217
m m
I
i
0-5
49 Age,
days
!O flies/O-ImL
I f l y / O ' l rnL Oriqin
Fxo. 5. Comparison o f esterase banding patterns o f a concentrated extract f r o m young males w i t h a standard extract f r o m an old male.
eggs than were present at eclosion had piled up in the abdomens of the older females; and in fact, many eggs were seen on grinding up the older females for the enzyme assays. The males show no increase in weight over their lifetime, and in fact are of a fairly constant mean weight at all ages--with the curious exception of a c a . 18 per cent drop (compared with the mean value for all the other age groups) in body weight exhibited by 49 day-old males. T A B L E 5.
M E A N WEIGHTS OF FROZEN FLIES. EACH VALUE IS MEAN W E I G H T (PER FLY) I N m g ARRIVED AT BY W E I G H I N G 2 0 FLIES TOGETHER
Age (days)
Females
Males
0.5 25 49 58 67
1"173 1"333 1.375 1.405 1.380
0-845 0.893 0.718 0.845 0.920
The assays of the two enzymes showed marked changes in activity as a function of age; and, again, the changes were different for the two sexes. G6PD activity (Fig. 6) for females rose almost threefold to a peak for 49 day-old flies, returning by day 67 to the initial (day 0.5) activity; though the standard deviations for most age groups are large, this rise in activity and subsequent fall is highly significant by t-tests (P < 0.005 for both the rise and the fall). Activity for this enzyme from males increased more than two fold, with a peak for 25 day-old flies, then sank to almost negligible activity for extracts from the oldest flies; again, both the rise and the fall are significant (P < 0.005). Alkaline
218
J . C. H A L L
9
i
x
~o 2O
/
g
/' \
",,I
____/(__ .....
0 5--
9,, I
I 25
] 49 Age,
[ 58
r 67
days
FIG. 6. Age-dependent changes in G6PD activity: mean AODs4o/min × 10.4 4- standard error of mean (standard deviation/~/n). A, Females; O, Males. Age (days) 0.5 25 49 58 67
Females 12 12 34 15 11
+ ± ± ± ~
1.0 1.2 3"8 2.6 2.3
Males 9 ± 0-8 23 ± 2-3 11 4- 0.9 7 ~ 0.3 3 + 0"6
phosphatase activity (Fig. 7) increased significantly (almost two-fold, P < 0.005) for the females, with a plateau for days 25 and 49, before falling to the level of initial activity by day 67. For the males, there was a greater than two-fold rise in activity to a peak for flies 49 days old. This increase is significant (P < 0.005) but the subsequent decrease for days 58 and 67 (to a level nearly 60 per cent higher than that for the youngest males) is not quite significant (P < 0.10). It should be noted that even if the weight increase for females (youngest vs. all older age groups) is taken into account, the changes which are seen are still highly significant. DISCUSSION Both the electrophoretic and the quantitative enzyme assays reveal patterns of agedependent changes which are markedly different for the two sexes. Komma (1968) has shown that G6PD produced by young Drosophila males differs from that produced by young females in terms of electrophoretic migration, kinetics, and stability. Further, this difference persisted in mixed homogenates of males and females, implying that something more than the temporary binding of small molecules is involved. Thus there may be fundamentally different physiological conditions prevailing in males and females with respect to enzyme synthesis. Such differences might account for the very different
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A 4OO -
MELANOGASTER
219
-
jl II
\\\
30O
0 x
I/
c
E
iiii
o 200
~'\ \ \\\
f
tm o
<3
iOC
0.5
1
i
25
49 Age,
i
58
c~7
doys
FIQ. 7. Age-dependent changes in alkaline phosphatase activity: mean AOD410/min × 10 -4 ± standard error of mean (standard deviation/~/n). A, Females; 0, Males. Age (days) 0"5 25 49 58 67
Females 169 335 318 228 175
i ± ~ ± ±
10"6 38"9 31"8 30-0 11"8
Males 107 116 219 180 164
~ ± i ± ±
9"6 8"4 40"4 25"7 28"0
N.B. For Fig. 6 and for Fig. 7, n = 24 flies for 0"5 day-old females and males; and n = 12 flies for females and males from all other age groups. Each point is a mean value :k one standard error of mean, resulting from 24 single-fly assays for 0" 5 day-old flies, and 12 such assays for flies from all other age groups. patterns of enzyme change with age that have been shown here. And these sex-specific enzyme changes might themselves be reflected in the different survival curves shown by males and females; that is, genetically influenced senescence might be mediated b y rather different processes in male and female Drosophila. A question arises as to whether the age-dependent peaks of enzyme activity result from m o r e enzyme being made or f r o m a more active enzyme being made in the same a m o u n t as for young individuals. Schneiderman, Young, and Childs (1966) feel that Drosophila alkaline phosphatase is a family of enzymes whose m e m b e r s are organ- or even tissue-specific. Perhaps a different, m o r e active alkaline phosphatase is made in flies 49 days old than is m a d e in newly emerged adults; and perhaps such a switch is intimately involved in senescence. T h e results reported here do point the way toward some questions as to how an organism's changing biochemical constitution might be
220
j . c . HALL
related to the process of senescence. Butit is clear that one cannot use only these survival curves and the results of the several assays meaningfully to construct specific, detailed models on the causes and mechanisms of aging. The purpose of the investigation has, however, been fulfilled: some objective, fairly precise symptoms of aging fruit flies have been established; at this point is is not crucial to causally relate these enzyme changes to a precise mechanism of senescence. But there is now some basis for trying to find possible "aging mutants": genetic variants of D. melanogaster which show patterns of enzyme changes different from those established here. One could, for example, mutagenize chromosomes or collect them from nature, make them homozygous, and then ask if there was induced or if there existed in nature a mutation whose phenotype is--with respect to the wild-type changes now established-repeatably different changes in enzyme banding patterns (earlier or later changes or ones that are altogether different in kind) and different patterns of increases and decreases in enzyme activity.
Acknowledgements--I should like to thank Drs. L. SANDLER,J. GALLANT and A. MOTULSKY for their guidance, comments and interest. I am also indebted to Drs. N. CARTER, J. GALLANT,and J. IRR for their expert technical instruction.
REFERENCES CASAR~rr, G. W. (1964) Adv. gerontol. Res. 1, 109. CHAPMAN,R. G., Hm~NmSEY, M. A., WALTERSDORPH,A. M., HUP-~EKENS, F. M. and G~'u3to, B. W. (1962)j~. din. Invest. 41, 1249. DEMoss, R. D. (1955) In Methods in Enzymology, (Edited by COLOWICK, S. P. and KAPL~"L N. O.) Vol. 1, p. 328. Academic Press, New York. ECHOLS, H., G ~ N , S. and TORRIP~'~I, A. (1961) J. mol. Biol. 3, 425. EPSTEIN, C. J., MARTIN, G. M., SCHULTZ, A. L. and MOTULKSY, A. G. (1966) Medicine 45, 177. GONZALEZ, B. M. (1923) drner. Naturalist 57, 289. HARRIS, H., HOPKINSON, D. A. and ROBSON, E. B. (1962) Nature, Lond. 196, 1296. KOMMA, D. J. (1968) Biochem. Genetics 1, 337. ROCKSTmN, M. (1966) In Topics in the Biology of Aging (Edited by KROHN, P. L.) p. 28. Interscience Publishers, New York, London and Sydney. SCHNEIDERMAN,H., YOUNG, W. J. and CHILDS,B. (1966) Science 151, 461. SMITHIES, O. (1955) Biochem. J. 61, 625. SZlLARO, L. (1959) Proc. Natl..4cad. Sci. U.S. 45, 30. S u m m a r y - - 1 . Marked age-dependent changes in four enzymes were found by assaying crude extracts of Drosophila melanogaster. A. Hexose-P-isomerase from very old females lacked two of the five bands characteristic of young female extracts run on starch gels; the staining intensity increased for the older females and for older males. B. Esterase from old males showed several additional electrophoretic bands which did not appear in assays of young males; in addition there was a large increase in staining intensity for male esterase and a lesser such increase for females. C. G 6 P D activity--quantitatively determined---showed a significant increase and then a decrease as a function of age; peak activity of the male enzyme was for flies 25 days old, while this peak appeared at 49 days for females. D. Alkaline phosphatase activity showed similar significant increases and decreases with age, with a plateau of activity occurring at 25 and 49 days for females, and a peak appearing with respect to males 49 days old. 2. Survival curves for flies kept in shell vials were determined. T h e y show later mortality for virgin females than for females who mate. Mated females had a slightly longer lifespan than mated or unmated males.
AGE-DEPENDENT ENZYME CHANGES IN D R O S O P H I L A
MELANOGASTER
3. These symptoms of aging Drosophila were established to form a basis for looking for, then recognizing, genetic variants whose phenotype is an altered pattern of senescent change. Characterization of such "aging mutants" could provide ways of understanding the process of senescence. R 6 a u m 6 - - 1 . Des alt6rations marqu6es, en fonction de l'age, de quatre enzymes ont 6t6 relev6es au titrage d'extraits crus de Drosophila melanogaster. A. L'hexose-P-isom6rase de tr~s vieilles femelles 6tait priv6e de deux des cinq bandes caract6ristiques des extraits de femelles jeunes port6s sur des gels d'amidon; l'intensit6 de coloration augmentait dans le cas des males et des femelles plus ag6s. B. L'est6rase de mMes ag6s pr6sentait ~ l'61ectrophor~se plusieurs bandes additionnelles, qui ne se manifestaient pas lors des titrages d'extraits de jeunes males; en outre, l'intensit6 de coloration augmentait fortement dans l'est6rase des males et beaucoup moins dans celle des femelles. C. D&ermin6e quantitativement, l'activit6 de la G6PD manifestait une augmentation significative, puis une diminution en fonction de l'age; le pic d'activit6 de l'enzyme se situait chez les mouches males ~ l'age de 25 jours, mais ne se manifestait qu'h 49 jours chez les femelles. D. L'activit6 de la phosphatase alcaline a manifest6 de semblables augmentations et diminutions significatives avec l'age, avec u n plateau d'activit6 A 25 et 49 jours pour les femelles et u n pic ~ 49 jours chez les males. 2. Les courbes de survie des mouches gard6es en flacons ont 6t6 d&ermin6es. Elles r6v~lent une mortalit6 plus tardive chez les femelles vierges que chez les femelles accoupl6es. La long6vit6 de ces derni~res &ait 16g6rement sup6rieure que celle des males, accoupl6s ou non. 3. Ces sympt6mes du vieillissement chez Drosophila ont 6t6 relev6s pour constituer une base d'examen, puis d'identification de viarantes g6n&iques dont le ph6notype est u n ensemble alt6r6 de modifications s6nescentes. La caract6risation de ces "mutants vieillissants" pourrait ouvrir des voles h la compr6hension du processus de s6nescence. Z u s a m m e n f a s s u n g - - 1 . I n Rohextrakten yon Drosophila melanogaster wurden ausgepr~igte altersabhiingige Ver~inderungen bei vier Enzymen gefunden. A. Bei Hexosephosphatisomerase von sehr alten Weibchen fehlten in Stiirkegelliiufen zwei der fiinf Banden, welche charakteristisch fiJr junge Weibchen sind. Die Anfiirbbarkeit stieg bei den iilteren Weibchen und Miinnchen. B. Esterase alter M~innchen zeigte mehrere zusiitzliche elektrophoretische Banden, die bei jungen Miinnchen nicht zu finden waren. AuBerdemstieg die Anffirbbarkeit bei Esterase von Miinnchen stark an, weniger stark bei Weibchen. C. Die quantitativ bestimmte G6PDH-Aktivitiit zeigt in Abhangigkeit vom Alter zun~ichst einen deutlichen Anstieg, dann einen Abfall. Der Gipfel lag bei m~nnlichen Fliegen beim 25. Lebenstag, bei Weibchen bei 49 Tagen. D. Die Aktivitiit der alkalischen Phosphatase zeigte iihnliche altersabhiingige Anstiege und Abnahmen. Ein Plateau trat bei Weibchen bei 25 u n d 49 Tagen auf. Bei Miinnehen lag der Gipfel am 49. Lebenstag. 2. Fiir in Schalenfliischen gehaltene Fliegen wurden die l~bedebenskurven bestimmt. Fiir unbefruchtete Weibchen ist die Lebensdauer grOBer als f'dr befruchtete. Befruchtete Weibchen hatten etwas l~ngere Lebensdauer als M~mchen. Die gilt gleichermaBen fiir gepaart habende u n d fiir nicht gepart habende Miinnchen. 3. Diese Befunde bei alternden Drosophila sollen eine Basis fiir die Suche u n d Erkennung yon genetischen Varianten mit ver~_nderten Phiinotyp der Altersveriinderungen bieten. Die Charakterisierung solcher "Alternsmutanten" k6nnte zum Verst~ndnis der Altersprozesse beitragen.
221
222
j . c . HALL P e ~ m M e - - 1 . [ I p H a u a J m 3 e Heo6pa6oTammLX 3KcTpaKTOB H3 Drosophila melanogaster 6 b L ~ O6Hapy'~eHH 3aMeTH~e H3MeHeHI.DI, 3aBHcsn~He OT Bo3pacTa, B qeT~pCx [~pMeHTaX. A . B r c K c o 3 e - - - P - - H 3 0 M e p a 3 e H 3 0 q e a b cTapI~x CaMOK a e ~ o c T a B a n o ~I~yx H3 I]~qTH IIOJIO¢, x a p a x T ~ p l ~ i X ~lJI~l3KCTpaKTOB 143 MOJIO]IbIX caMoK IIpH HCIIMTaHHH Ha KpaxMaJibm i x re~-~x; y 6oHee cTapbIx CaMOK H CaMlleB y s e m o m ~ a c s HHTeHCHBHOCTb oKpacKH. B. ~ T e p a 3 a H3 cTapsIX CaMIIOB BbLqBHHa HOCKOHbKO ROHOJIHHTenbHbI X3HeKTpo~bopeTHqeCKHX IIOJIOC, KOTOpMe He O6Hapy3KHBaJIHCI, npH aHa~H3e y MO~O~bLX CaMUOS; KpOM¢ TOrO, 6ht~O 6o~IbmOe noBbimeHHe B HHTeHCTIBHOCTH oKpacKH 3cTepa3bx CaMUOB H M~H~ B~ipamem~oe n o e b l m e t m e y caMoK. B. AKT~SHOCTb r m o x o 3 b x 6-~boc0aTa ~lerm~poreHa3m ( G 6 P D ) - - n p H ~Om~e~rBeHHOM o n p e ~ e a e m m m, m s s n a c H a q a ~ a 3Ha~mTemsHoe n o s b ~ m e t m e , a 3aTOM CHH~eHHe C s o z p a c TOM; BbICl~a.q T O g a aKTHBHOCTH ~pepMeHTa CaMHOB o 6 H a p y m t m a n a c b y Myx B e o 3 p a ~ r e 25-H ~Hett, T o r ~ a Ka~ y caMoK rm~ HO~BJLqHC~ ~ Bo3pacTe 49-H ~Hett. ~ . Axw~m~OCWb m e n o ~ m o a ~boc~bawa3bi o 6 H a p y m H s a ~ a CXO~HSIe 3Ha~HTeJIbSble I~OBbImeHmq H CHH~¢HH~ C eo3pacTOM, npH~¢M BbICHIH~ y p o e e m , aKTHBHOCTH y CaMO~ IlOCTHraJICll B Bo3pacwe 25--rt H 49--H ~I8el:i, a y caMraoa a Bo3pacTe 4 9 - r t ./~Hel~. 2. Onpe/Iemumc~, xprem],ie m,~acnaaeMocTrl ~ n a Myx rlpH co~lep~armH B paKOBHHH],~X c ~ q n K a x . O n n o6Hapyr, o t r m 6 o n c e noa,aHrOIO CMepTHOC'rb y ,KeBCTBOHHblX CaMOK, qeM y c n a p H a a r o m H x c ~ CaMOK. ~ cnapeHHMX CaMOK ~'IHT~JIbHOCTb ~EH3HH 6 u n a HeCKOJIbI(O BbIme, qeM y cnapenHbLX H HecHapeHH/~IX CaMI~OB. 3. ~TH CHMIITO1VEbl cTapeHHI/ y Drosophi[~l ~bIfIH yCTaHOB~IeHI~I KaK OCHOBa~ HCXO~$I H3 KOTOpOI~ MO3KHO HCKaTb~ a 3aTeM pacIIO3HaTb, rCHeTI~eCKHe BapHaHTbI, (I)eHOTHH KOTOpbIX npe~CTaBJDIeT CO~Oi~ BH~OH3MCHeHHe THHa H3MeHeHH~ HpH CTapeHHH. XapaKTepH3al~H~l TaKHX "CTapetoI/~HX MyTaHTOB" MO~I£eT BbIIIBHTb ~aHHble ~rLq HOHHMaHH~I H p o u e c c a cTapCHH~I.